PROJECT SUMMARY Electrical stimulation (ES) of low-threshold A?-nerve fibers is a clinical strategy for treating chronic pain that is refractory to pharmacotherapies, including opiates. However, the mechanisms underlying pain inhibition by A?-fiber activation remain elusive, limiting further clinical and technological improvements. Our major goal is to uncover spinal neuronal and non-neuronal mechanisms of pain inhibition by A?-ES, and to identify targets for rationally selecting adjuvant drugs to enhance pain inhibition and avoid side effects. In Aim 1, by using animal models of neuropathic pain, we will first delineate spinal neuronal mechanisms by which A?-ES inhibits pain transmission. Electrophysiology studies will uncover how different forms of A?-ES modulate functionally distinct subsets of superficial dorsal horn neurons (e.g., inhibitory vs. excitatory neurons), and further differentiate the underlying receptor mechanisms. We postulate that the excitatory neuron-preferred inhibition may enable A?-ES to be used together with low-dose adjuvant drugs for circuitry-specific enhancement of pain inhibition. We will test whether limiting endogenous adenosine degradation or GABA reuptake during A?-ES specifically enhances the inhibition of dorsal horn excitatory and projection neurons and increases the net inhibition of pain transmission. In Aim 2, we will then unveil a non-neuronal pain gating mechanism activated by A?-ES. By conducting high-throughput GCaMP6 imaging and electrophysiology recording, we will examine whether A?-ES activates spinal astrocytes to induce inhibition of spinal nociceptive transmission, and whether this process is attenuated by nerve injury. Since pain inhibition by dorsal column stimulation (DCS) is intrinsically linked with activation of A?-fibers, DCS will be used as a proof-of-principle for studying A?-ES in vivo. We will examine whether DCS changes astrocyte reactive markers, affects the levels of pro-inflammatory mediators, and promotes microglial polarization from M1 to M2 state after nerve injury. We will further test whether glia-derived adenosine inhibits excitatory or projection neurons and enhances A?-ES?induced inhibition of these neurons. Our findings in Aims 1 and 2 will help us to develop new strategies of enhancing pain inhibition by A?-ES in Aim 3. We will test whether inhibition of neuropathic pain-related behavior by DCS can be enhanced by limiting degradation of endogenous adenosine and inhibiting GABA reuptake in the spinal cord. Owing to use-dependent and circuitry-specific features, we expect that the treatment will not induce side effects. Our findings will reveal new mechanisms underlying pain inhibition by A?-ES, and will provide important rationales and drug targets for future translational studies aimed at using low-dose adjuvant drugs to improve the efficacy and specificity of pain inhibition by A?-ES therapies.